Generation And Functional Analysis Of Novel Monoclonal Antibodies To VWFA2 Domain That Inhibit Interactions Between VWF And ADAMTS13

Part I Production and characterization of novel monoclonal antibodies against VWF domain A2Objective: The plasma von Willebrand factor(VWF)is a large multimeric glycoprotein that promotes the tethering of platelets to sites of vascular injury and plays a critical role in hemostasis.The adhesive activity of VWF is positively correlated with the size of VWF multimers in plasma.ADAMTS13(a disintegrin and metalloproteinase with a thrombospondin type 1 motif,member 13)regulates the multimeric size of von Willebrand factor(VWF)by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain.A minimal region as a functional substrate forADAMTS13 consisted of 73 amino acid residues from D1596 to R1668 of VWF,designated VWF73.Deletion of residues E1660-R1668 of the C-terminal ?-helix within VWFA2 abrogates cleavage by ADAMTS13.This indicates that the C-terminal ?-helix of VWFA2 is required forADAMTS13 to bind and cleave VWF.Preparation and epitope mapping of murine monoclonal antibodies(mAbs)directed against the C-terminal ?-helix of human VWFA2 may offer a new tool for further research on the reactions between VWF and ADAMTS13.Methods: Balb/c mice were immunized with a polypeptide consisting of VWF residues R1659-R1668.mAbs were developed by standard hybridoma technology and identified by enzyme-linked immunosorbent assay(ELISA).The epitopes of mAbs were mapped using recombinant VWFA2 and a series of its truncated and deletion mutants with western blotting analysis.Results: We obtained six hybridoma clones producing anti-VWFA2 mAbs,with clone numbers 5D8,8F1,9D6,9G11,9A11 and 9C11.One mAb(9G11),designated SZ-179,was found to have high affinity(50 ng/ml)for a synthetic peptide encompassing residues1659–1668 and native VWF.SZ-179 was identified as an Ig G1 subtype by ELISA.SZ-179 reacted specifically with recombinant VWFA2 and GST-VWF73-His,respectively,but not with recombinant VWFA1 or VWFA3.Epitope mapping experiments revealed that SZ-179 bound to the distal portion of the VWF A2 domain(E1660-L1666).Conclusions: SZ-179 is a novel mAb to the A2 domain of VWF,which can be used as a tool for further study of mechanism of interactions between VWF and ADAMTS13.Part II Functional analysis of SZ-179Objectives: We determined whether mAb SZ-179,which is against the C-terminal?-helix of human VWFA2,affects the susceptibility of VWF to proteolysis by ADAMTS13.Methods: The effect of SZ-179 to GST-VWF73-His on its proteolysis by recombinant ADAMTS13(rADAMTS13)was measured by 15% SDS-PAGE under reducing condition and western blotting analysis.The effect of SZ-179 to VWF or VWF-R1597 W on its proteolysis by rADAMTS13 were investigated under static/denaturing or native conditions by 1.3% agarose gel electrophoresis and immunologic analysis.Results: SZ-179 inhibited proteolytic cleavage of GST-VWF73-His by rADAMTS13 in a concentration-dependent manner,with a half maximal inhibitory concentration(IC50)of 221.8 ?g/ml.SZ-179 also reduced degradation of high molecular-weight(HMW)-VWF-multimers under static/denaturing condition dose-dependently,with an IC50 of 0.66 ?g/ml.Similarly,the decreased amount of the high and intermediate molecular weight multimers of R1597 W mutant were dramatically reduced by SZ179 in a concentration-dependent manner under native condition(IC50 13.54 ?g/ml).Conclusions: SZ-179,a novel mAb to the A2 Domain of VWF,inhibits theinteraction between VWF and ADAMTS13,and prevents excessive degradation of HMW-VWF multimers.This mAb may be useful for the development of therapeutic options to treat bleeding episodes.